ICP0-mediated enhanced expression system

ABSTRACT

Methods and compositions for increasing the production of recombinant proteins by introducing ICP0 to cells capable of producing a recombinant protein are encompassed. In one method, the recombinant protein is a protein that is required for the replication of a replication defective virus, wherein the recombinant protein is provided to the replication defective virus in trans.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a national phase entry pursuant to 35 U.S.C. § 371of International Application No. PCT/US2016/032082, filed May 12, 2016,which claims the benefit of priority of U.S. Provisional Application No.62/161,194, filed May 13, 2015, which are incorporated by reference intheir entirety for any purpose.

FIELD

Methods and compositions for enhancing expression of recombinantproteins are provided.

BACKGROUND

The efficient expression of recombinant proteins is essential forproduction of therapeutic proteins, particularly when the protein ispartially toxic to the host cell producing it. Efficient expression isalso vital when producing replication defective virus for use invaccines. In this scenario, certain essential genes are removed from theviral genome to prevent viral replication in the vaccine. However, inorder to grow the vaccine virus in cell culture, the proteins requiredfor replication of the virus may be provided in trans to complement thereplication defective virus, and suboptimal expression of thesecomplementary proteins may limit viral yield.

Genital herpes is a sexually transmitted disease usually caused byinfection with herpes simplex virus type 2 (HSV-2). After infection,HSV-2 can persist with latent virus in the neural ganglia, allowingepisodic outbreaks of painful genital lesions in up to 25% of patientswho are infected. Transmission of HSV from an infected woman with activedisease to her newborn can also lead to severe neurologic complicationsor death in the baby.

The life cycle of HSV can be divided into lytic infection ofkeratinocytes and fibroblasts and latent infection of sensory neurons.During HSV's latent stage, its genome remains quiescent with few or nogenes expressed. External stressors may activate HSV's lytic phase,where a full program of gene expression and genome replication isactivated yielding new virions.

As with many viruses, HSV genes can be divided into three broadcategories, called immediate early (IE), early (E), and late (L),depending on the timing of their expression after infection. Herpessimplex virus infected cell polypeptide zero (“ICP0”) is an IE proteinfound in HSV and related alphaherpesviruses. ICP0 was discovered in thelate 1980s and early 1990s, but its function is still largely unknown.It has been reported that ICP0 may activate gene transcription, anddeletion of ICP0 reduces HSV replication in vitro and attenuates thevirus in vivo by inactivating its ability to transition to lyticreplication. See, e.g., Boutell and Everett, J. Gen. Virol (2013)94:465-481, and Lanfranca et al, Cells (2014) 3:438-454.

At the molecular level, ICP0 has a highly complex phenotype. At leastfour mechanisms have been proposed to explain how ICP0 modulates genetranscription. These generally involve the modification of chromatin inorder to de-repress the HSV genome after replication in neuronal cells.ICP0 has also been proposed to interact with transcription factorE2FBP1. In addition, a fundamental feature of ICP0 is its E3 ubiquitinligase activity mediated by its RING domain. ICP0 causes ubiquitinationof proteins, which leads to their degradation or changes in theirfunction. A number of proteins targeted for ubiquitination by ICP0participate in mechanisms through which cells resist viral infections.For example, PML, a component of the ND10 nuclear bodies, whichinterfere with HSV genome replication, is degraded by ICP0-mediatedubiquitination, as are other proteins such as IFI16 and IkBa involved ininnate immunity to infections. Despite the apparent importance of ICP0in HSV life cycle, very little is known about ICP0.

Development of an HSV-2 vaccine may be able to prevent herpes diseaseand block spread of the virus. Previous clinical studies of an HSV-2subunit vaccine containing glycoprotein D, however, failed to showefficacy at preventing HSV-2 infection, see R. B. Belshe et al., NEJM366:34-43 (2012). It has been proposed that a vaccine capable ofmimicking natural viral infection and inducing a broad immune responsemay be an attractive vaccine candidate, as noted in M. C. Bernard etal., PLOS One 10(4):e0121518 (2015).

HSV529 is a replication defective HSV-2 variant, also known as ACAM529,as described in Delagrave et al, PLOS One 7(10):e46714 (2012), and M. C.Bernard et al., PLOS One 10(4):e0121518 (2015). The HSV529 virus, whichis a plaque-purified clone of the re-derived strain d15-29 lacks twoviral DNA replication genes (UL5 and UL29) and cannot replicate in theabsence of these gene products. Da Costa et al. J. Virol. 7963-7971(2000); S. T. Mundle, PLOS One 8(2):e57224 (2013). The HSV529 virus isgrown using the complementary helper cell line, AV529-19, which is aVero cell line that was stably transfected to supply UL5 and UL29 HSV-2proteins in trans in infected cells. In mouse and guinea pig models ofgenital herpes, HSV529 induces immune responses and blocks HSV-2infection, see M. C. Bernard et al., PLOS One 10(4):e0121518 (2015) andreferences therein.

We herein describe that the wild-type HSV-2 virus generatessignificantly higher virus yield than the HSV529 after infection ofAV529-19 cells. Given the need to generate the HSV529 clinical candidateat a large scale for clinical purposes, we investigated methods toincrease viral yield. Given the known critical role of ICP8 (the proteinexpressed by the U_(L)29 gene) in HSV-2 replication, see P. E. Boehmeret al., J Biol Chem 269(46):29329-3 (1994), we investigated expressionof ICP8 in AV529-19 cells. We unexpectedly found that ICP8 could only bedetected in AV529-19 cells after infection with HSV529. Thus, thereexists a need in the art to improve recombinant protein production inAV529-19 cells to increase the yield of HSV529 production. There is alsoa need in the art to increase protein production in other systems, suchas, for example, when producing therapeutic biologics for massproduction, and when producing replication defective vaccine in transcomplementary systems.

SUMMARY

In evaluating the means by which ICP8 is increased in AV529-19 cellsfollowing HSV529 infection, we have determined that the immediate earlyHSV protein, ICP0, is capable of inducing AV529-19 cells to increaseexpression of ICP8. Delivery of recombinant ICP0, by transduction ortransfection, can increase levels of ICP8, as well as being able toincrease expression of recombinant proteins. In addition, expression ofICP0 in AV529-19 cells before infection with the HSV529 virus leads tosignificantly increased viral yields.

In accordance with the description, the inventors have achievedincreased expression of proteins through co-expression of ICP0. In oneembodiment, a method for increasing the yield of recombinant proteinexpression in vitro is encompassed, wherein ICP0 is introduced to a cellexpressing a recombinant protein, thereby increasing the yield of therecombinant protein. In one embodiment the cell is AV529-19, and therecombinant protein is ICP8 or pUL5.

In one embodiment, the invention comprises a method for producingreplication defective virus or viral vaccine in culture. The methodcomprises providing a cell comprising at least one recombinant protein,wherein this recombinant protein is required for the replication of anotherwise replication defective virus. Introduction of ICP0 to the cellis done to increase expression of the recombinant protein, thereplication defective virus is introduced to the cell, and thereplication defective virus or viral vaccine is isolated. Theintroduction of ICP0 increases the yield of the replication defectivevirus.

In certain embodiments, the cell is AV529-19, and the recombinantprotein is ICP8 or pUL5.

In certain embodiments, the recombinant protein complements areplication defective virus in trans.

In another embodiment, the replication defective HSV-2 vaccine HSV529 isproduced in AV529-19 cells after introduction of ICP0. ICP0 isintroduced to AV529-19 cells, wherein ICP0 increases the expression ofat least one of ICP8 or pUL5 complementing proteins. AV529-19 cells areinfected with the HSV529 virus, and the HSV529 vaccine is isolated. Theintroduction of ICP0 increases the yield of the replication defectiveHSV-2 vaccine HSV529.

In certain embodiments, the nucleic acid encoding the recombinantprotein is episomal or integrated into the host cell genome. Thisnucleic acid may be DNA or RNA.

ICP0 may be introduced to the cell by transfection or transduction. ICP0may be introduced by transduction with HSV-2, replication defectiveHSV-2, HSV529, or HSV-1. ICP0 may also be introduced by transductionwith an adenovirus expressing ICP0. ICP0 may be introduced bytransfection with a plasmid encoding ICP0; this plasmid may have aninducible or non-inducible promoter that drives expression of ICP0. Theinducible promoter may be selected from chemically- orphysically-regulated promoters. Expression of ICP0 by this induciblepromoter may be regulated by a small molecule, such as doxycycline. Thenon-inducible promoter may be selected from the group consisting of CMVand SV40 promoters, as well as those containing known promoter elementssuch as TATA box, GC-box, CCAAT box, B recognition element, andinitiator element.

In certain embodiments, the quantity of recombinant protein produced orisolated is greater than the quantity of recombinant protein produced orisolated in control cells that do not comprise ICP0.

Additional objects and advantages will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice. The objects and advantageswill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the claims.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description, serve to explain the principles described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows growth of HSV529 when it is added to a monolayer of cellscomprising both the UL29 and UL5 complementing genes. The plates on theright side show monolayers of the Vero lines AV529-19 (upper right) andV529 (lower right), both of which express both UL5 and UL29. V827 cells(upper left) express UL29 but not UL5; while L2-5 cells (bottom left)express UL5 but not UL29.

FIG. 2 shows increased replication of wild-type HSV-2 compared to HSV529in AV529-19 cells. AV529-19 cells were infected with either HSV529 orwild type HSV-2, and infected cells were harvested and lysed. Titers ofHSV-2 were then determined by plaque assay on complementing AV529-19cells. P=0.0173, by 2-tailed t-test.

FIG. 3 shows induced expression of ICP8 when AV529-19 cells are infectedwith HSV529. AV529-19 cells were either mock infected (NC) or infectedwith HSV529 and then allowed to incubate for 41 hours under standardcell culture conditions. Cells were harvested and lysed in SDS-PAGEsample buffer, and the resulting samples were analyzed by westernblotting.

FIG. 4 shows induction of ICP8 expression encoded by a repliconfollowing HSV529 infection. Western blots were performed usingchemiluminescence following the induction of ICP8 expression by d15-29(a.k.a., HSV529) in AV529-19 cells (labeled as AV529) and in Vero cellstransfected with a VEE replicon expressing UL29 or UL5. The numbering atthe top of the blot indicates hours post-infection; the numbering on theleft indicates molecular weight.

FIG. 5 shows recombinant UL29 mRNA concentrations after infection withHSV-2 or HSV529. Taqman quantitative reverse transcription PCR (qRT-PCR)was used to measure mRNA in either unmodified Vero cells or AV529-19cells following infection with either HSV-2 (strain 186 syn+1) or HSV529at 0, 6, 24, 48, and 72 h post-infection. The qRT-PCR primers and probewere specific for the 3′ UTR of the pcDNA3.1+ vector used to expressrecombinant UL29 in AV529-19 cells. Error bars show standard deviation.

FIG. 6 shows increased expression of ICP8 by transduction of ICP0 in thecomplementing cell line AV529-19. AV529-19 cells were transduced withthe indicated adenovirus vector (MOI=25) and harvested 24 h later.

FIG. 7 shows induction of ICP8 by ICP0 infection in AV529-19 cells. Thecell line HEK293A, a complementing cell line used to produce adenovirusvectors, and the AV529-19 cell line were infected with an adenovirusvector expressing ICP0. Infected cells were harvested 24 h afterinfection and analyzed by western blotting.

FIGS. 8A and 8B show that co-transduction of ICP0 increases theexpression of episomally encoded recombinant proteins. AV529-19 cellswere transduced with adenovirus vectors expressing gD313, VP16, orluciferase (Luc) and then 24 h later with either an empty adenovirusvector or an adenovirus expressing ICP0. Twenty-four hours afterco-transduction, samples were collected for western blots.

FIGS. 9A and 9B show that ICP0 does not alter host cell proteinexpression patterns. Cells were co-transduced with the indicatedadenovirus vectors and with either an empty vector or with a vectorexpressing ICP0, as shown respectively by the − or + symbols. In FIG.9A, cell lysates were harvested 24 h post-transduction and analyzed bywestern blotting against the housekeeping protein GAPDH. In FIG. 9B,cell lysates were analyzed by SDS-PAGE and Simple-Blue staining.

FIG. 10 shows that ICP0 enhances CMV or SV40 promoter-driven expressionof plasmid-encoded genes. AV529-19 cells were transiently transfectedwith plasmids and incubated for 24 h. The plasmids used, pGL4.50 andpGL4.13 (Promega), encode firefly luciferase under the control of theCMV and the SV40 promoter, respectively. The transfected cells were thentransduced with a control adenovirus vector or a vector expressing ICP0.Cells were harvested 36 h later and analyzed by luminescence assay.

FIG. 11 shows enhanced expression of plasmid-encoded genes in Vero cellsinduced by ICP0. Vero cells were transiently transfected with plasmidsand allowed to recover for 24 h. These plasmids encoded either notransgene (Ctrl plasmid) or firefly luciferase under the control of theCMV promoter (CMV-Luc). The cells were then transduced with a controladenovirus (Ad) vector or a vector expressing ICP0 (Ad ICP0). Cells wereharvested 36 h later and analyzed by luminescence assay. P<0.0001 forthe difference between control adenovirus vector and adenovirusexpressing ICP0; error bars show one standard deviation.

FIG. 12 indicates that non-conservative ICP0 mutants do not induce ICP8expression in AV529-19 cells. AV529-19 cells were transduced withadenovirus vectors comprising no foreign gene (AdVect), wild type ICP0(AdICP0), a mutant of ICP0 in which the N-terminal approximately ⅓ isdeleted and the C-terminus is labeled with a poly-histidine tag (⅔ ICP0C-H), or a mutant of ICP0 in which approximately 5% of all amino acidshave non-conservative substitutions and a C-terminal poly-histidine tag(ICP0 5% mut-H). Non-transduced cells (NC) were also included asnegative controls. Following either 48 or 24 h of incubationpost-transduction, as indicated, cells were harvested and analyzed bywestern blotting with a rabbit polyclonal anti-HSV-2 antibody.

FIG. 13 shows that plasmid transfection of ICP0 increases recombinantprotein expression in AV529-19 cells. AV529-19 cells were either nottransfected (NC), transduced with an adenovirus expressing ICP0(AdICP0), transfected with the empty vector pcDNA3.1 (pcDNA),transfected with ICP0 expressed from the plasmid pJ603 (ICP0 wt-pJ603),or transfected with ICP0 with a C-terminal poly-histidine tag (ICP0wt-His-pcDNA). Forty-two hours post-transfection, cells were harvestedand analyzed by western blotting. The top blot was probed with therabbit anti-HSV-2 polyclonal antibody, which cross-reacts with HSV-1ICP8 (arrow), while the lower blot used the same samples and was probedwith a mouse anti-HSV-1 ICP8 monoclonal antibody (arrow).

FIG. 14 shows that the adenovirus vector itself does not contribute tothe ICP0-mediated enhanced expression effect. AV529-19 cells weretransfected with the indicated plasmids, and 24 h later eithertransduced with an adenovirus vector (+) or not transduced (−). Controlcells were left untransfected and later transduced with adenovirusexpressing ICP0 (Ad ICP0). Samples were harvested 36 hpost-transduction, and western blotting analysis of the cells wascarried out using an anti-HSV-1-ICP8 mouse monoclonal antibody.

FIG. 15 shows that ICP0 transduction increases the yield of HSV529 inAV529-19 cells. AV529-19 cells were transduced with an empty adenovirusvector (Ad vector) or an adenovirus vector expressing ICP0 (Ad ICP0) andwere infected with HSV529 either 1 h, 4 h, or 22 h later. One sample ofcontrol cells was not infected with adenovirus (HSV529 only), providinga baseline for HSV529 yield. Other samples were transduced with Advector or Ad ICP0 4 h prior to a mock infection instead of beinginfected with HSV529. Cells were harvested 24 h after infection or mockinfection with HSV529, and lysate was analyzed by plaque assay toquantify production of HSV529.

FIG. 16 shows induction of ICP8 expression by doxycycline using adoxycycline-sensitive vector to express ICP0. AV529 cells weretransfected with the pTetOne vector (Clontech), or engineered versionsof the plasmid which express ICP0 (TetOne-ICP0) or an ICP0 to which isappended a polyhistidine tag at the carboxyl terminus (TetOne-ICP0 his).Transfected cells were incubated in the presence of 0, 0.1, or 1 μg/mLof doxycycline, harvested, lysed, and analyzed by western blotting usingan anti-HSV-2 polyclonal rabbit antibody that detects ICP8.

DESCRIPTION OF THE SEQUENCES

Table 1 provides a listing of certain sequences referenced herein.

TABLE 1 Description of the Sequences SEQ ID Description Sequences NOForward GCCAGCCATCTGTTGTTTGC 1 primer of 3′UTR sequence of pcDNA3.1used to transfect AV529-19 Reverse GGGAGTGGCACCTTCCA 2 primer of 3′UTRsequence of pcDNA3.1 used to transfect AV529-19 Probe of CCCCGTGCCTTCCTT3 3′UTR sequence of pcDNA3.1 used to transfect AV529-19 HSV-1 ICP0MEPRPGASTR RPEGRPQREP APDVWVFPCD 4 strain 17RDLPDSSDSE AETEVGGRGD ADHHDDDSAS EADSTDTELF ETGLLGPQGV DGGAVSGGSPPREEDPGSCG GAPPREDGGS DEGDVCAVCT DEIAPHLRCD TFPCMHRFCI PCMKTWMQLRNTCPLCNAKI VYLIVGV1PS GSFSTIPIVN DPQTRMEAEE AVRAGTAVDF IWTGNQRFAPRYLTLGGHTV RALSPTHPEP TTDEDDDDLD DADYVPPAPR RTPRAPPRRG AAAPPVTGGASHAAPQPAAA RTAPPSAPIG PHGSSNTNTT TNSSGGGGSR QSRAAAPRGA SGPSGGVGVGVGVVEAEAGR PRGRTGPLVN RPAPLANNRD PIVISDSPPA SPHRPPAAPM PGSAPRPGPPASAAASGPAR PRAAVAPCVR APPPGPGPRA PAPGAEPAAR PADARRVPQS HSSLAQAANQEQSLCRARAT VARGSGGPGV EGGHGPSRGA APSGAAPLPS AASVEQEAAV RPRKRRGSGQENPSPQSTRP PLAPAGAKRA ATHPPSDSGP GGRGQGGPGT PLTSSAASAS SSSASSSSAPTPAGAASSAA GAASSSASAS SGGAVGALGG RQEETSLGPR AASGPRGPRK CARKTRHAETSGAVPAGGLT RYLPISGVSS VVALSPYVNK TITGDCLPIL DMETGNIGAY VVLVDQTGNMATRLRAAVPG WSRRTLLPET AGNHVMPPEY PTAPASEWNS LWMTPVGNML FDQGTLVGALDFRSLRSRHP WSGEQGASTR DEGKQ HSV-2 ICP0 MEPRPGTSSR ADPGPERPPR QTPGTQPAAP5 strain HG52 HAWGMLNDMQ WLASSDSEEE TEVGISDDDLHRDSTSEAGS TDTEMFEAGL MDAATPPARP PAERQGSPTP ADAQGSCGGG PVGEEEAEAGGGGDVCAVCT DEIAPPLRCQ SFPCLHPFCI PCMKTWIPLR NTCPLCNTPV AYLIVGVTASGSFSTIPIVN DPRTRVEAEA AVRAGTAVDF IWTGNPRTAP RSLSLGGHTV RALSPTPPWPGTDDEDDDLA DVDYVPPAPR RAPRRGGGGA GATRGTSQPA ATRPAPPGAP RSSSSGGAPLRAGVGSGSGG GPAVAAVVPR VASLPPAAGG GRAQARRVGE DAAAAEGRTP PARQPRAAQEPPIVISDSPP PSPRRPAGPG PLSFVSSSSA QVSSGPGGGG LPQSSGRAAR PRAAVAPRVRSPPRAAAAPV VSASADAAGP APPAVPVDAH RAPRSRMTQA QTDTQAQSLG RAGATDARGSGGPGAEGGPG VPRGTNTPGA APHAAEGAAA RPRKRRGSDS GPAASSSASS SAAPRSPLAPQGVGAKRAAP RRAPDSDSGD RGHGPLAPAS AGAAPPSASP SSQAAVAAAS SSSASSSSASSSSASSSSAS SSSASSSSAS SSSASSSAGG AGGSVASASG AGERRETSLG PRAAAPRGPRKCARKTRHAE GGPEPGARDP APGLTRYLPI AGVSSVVALA PYVNKTVTGD CLPVLDMETGHIGAYVVLVD QTGNVADLLR AAAPAWSRRT LLPEHARNCV RPPDYPTPPA SEWNSLWMTPVGNMLFDQGT LVGALDFHGL RSRHPWSREQ GAPAPAGDAP AGHGE BICP0MAPPAAAPEL GSCCICLDAI TGAARALPCL 6 BovineHAFCLACIRR WLEGRPTCPL CKAPVQSLIH herpes virusSVASDECFEE IPVGGGPGAD GALEPDAAVI 1.1 strainWGEDYDAGPI DLTAADGEAS GAGGEAGAAD Jura GSEAGGGAGG AEEAGEARGA GAGRAAGAAGGRAGRGADAA QEFIDRVARG PRLPLLPNTP EHGPGAPYLR RVVEWVEGAL VGSFAVTARELAAMTDYVMA MLAECGFDDD GLADAMEPLI GEDDAPAFVR SLLFVAARCV TVGPSHLIPQQSAPPGGRGV VFLDTSDSDS EGSEDDSWSE SEESSSGLST SDLTAIDDTE TEPETDAEVESRRTRGASGA ARARRPAERQ YVSTRGRQIP AVQPAPRSLA RRPCGRAAAV SAPPSSRSRGGRRDPRLPAA PRAAPAAQAR ACSPEPREEG RGAGLGVAAG ETAGWGAGSE EGRGERRARLLGEAGPPRVQ ARRRRRTELD RAPTPAPAPA PAPAPISTVI DLTANAPARP ADPAPAAAPGPASAGAQIGT PAAAAAVTAA AAAPSVARSS APSPAVTAAA TSTAAAISTR APTPSPAGRAPAADPRRAGA PALAGAARAE VGRNGNPGRE RRPASAMARG DLDPGPESSA QKRRRTEMEVAAWVRESLLG TPRRSSAALA PQPGGRQGPS LAGLLGRCSG GSAWRQ

DESCRIPTION OF THE EMBODIMENTS

ICP0 stimulates recombinant protein expression in a manner that isindependent of the promoter used to drive the expression of therecombinant protein. ICP0 may be introduced to a cell comprising anucleic acid encoding a recombinant protein to increase the expressionof the recombinant protein. The nucleic acid may be episomal (encoded ona plasmid) or integrated in the host cell genome. ICP0 may be introducedvia transfection or transduction, and may be associated with aconstitutive or inducible promoter.

Replication Defective Viral Vaccines

Replication defective viruses are known to those skilled in the art tobe a means of developing vaccines, such as those for diseases notamenable to use of inactivated virus or live-attenuated virus vaccines(See T. Dudek and D. M. Knipe Virology 344(1):230-9 (2006)). Replicationdefective viruses lack one or more functions essential for replicationof the viral genome or synthesis and assembly of viral particles. Due tothe fact that replication defective viruses do not propagate in normalcells, they can be used as vaccines to present viral antigens andstimulate an immune response. Thus, there is a need to produce highyields of replication defective virus for use in immunizing againstcertain pathogens.

Encompassed in this invention are methods for producing replicationdefective virus, such as, for use as vaccines. In one embodiment thereplication defective virus is the clinical candidate HSV529. HSV529 isa replication defective virus for prevention of HSV-2 infection. Incertain embodiments, the replication defective virus includes thoseknown in the art. Non-limiting examples include members of the herpesfamily of viruses including, alpha herpes viruses, herpes simplex virustypes 1 and 2, and varicella-zoster viruses. This invention is notlimited to specific viruses, as the methods for generating a replicationdefective virus for any particular virus would be understood to thoseskilled in the art. The methods of producing replication defective viruscomprise providing a cell capable of producing a recombinant proteinwith ICP0, infecting the cell with the replicating defective virus(either after or at the same time as the ICP0), and isolating thereplication defective virus. The method may also comprise the furtherstep of purifying the isolated virus.

Similarly, methods for increasing the yield of replication defectiveviruses, such HSV529 are encompassed, comprising providing a cellcapable of producing a recombinant protein with ICP0, infecting the cellwith the replicating defective virus (either after or at the same timeas the ICP0), and isolating the replication defective virus, wherein theamount of virus isolated is greater than the amount of virus isolatedfrom a control cell system that did not receive ICP0. The method mayalso comprise the further step of purifying the isolated virus.

Complementary Cells Lines for Production of Replication Defective ViralVaccines

Complementary cell lines for production of replication defective viralvaccines express the missing viral gene product(s) and allow replicationof the defective virus (See T. Dudek and D. M. Knipe Virology January 5;344(1):230-9 (2006)). Replication defective viruses are unable toreplicate in normal cells, as one or more steps in viral replication areblocked. In certain embodiments, the complementary cell line can providethe defective viral proteins for propagation to the replicationdefective virus in trans, such that the specific viral proteinsexpressed by the complementary cell line can be used by the virus in itsreplication process to allow propagation of the virus. In oneembodiment, AV529-19 is a Vero cell line (ATCC, CCL-81.2), which hasbeen stably transfected to supply the UL5 and ICP8 HSV-2 proteins intrans that are needed for propagation of the HSV529 virus for generationof this vaccine against HSV-2 infection. AV529-19 cells are encompassedin the methods of the invention for use in complementing the replicationdefective virus HSV529.

Recombinant Proteins

Recombinant proteins are proteins made from DNA sequences that would nototherwise be found in biological organisms. In one embodiment, therecombinant protein is generated from recombinant DNA, wherein the DNAcomprises genetic material that is supplied from different sources(e.g., species) or from sequences that have been created that would nototherwise be found in biological organisms. Recombinant proteins may bemodified from the natural sequence or may have differences inpost-translational modifications. Recombinant proteins may also allowproduction of protein in cells that would not normally express it orallow production of large quantities of proteins. Recombinant proteinscan be viral or non-viral. Non-viral recombinant proteins can be derivedfrom mammalian, plant, insect, or other non-viral protein sources. Incertain embodiments, ICP8 or pUL5 may be the recombinant protein that isexpressed. In one embodiment, the ICP8 and pUL5 expressed are those ofHSV-2. In certain embodiments, the ICP8 and pUL5 are those of HSV-1 orother viruses.

Cells That Can Express Recombinant Proteins

In certain embodiments, cells that can express recombinant proteinsinclude mammalian cells, plant cells, insect cells, yeast cells,bacterial cells, avian cells, and others. In certain embodiments, thecell may be Vero, BHK, CHO, HKB, HEK, NSO, U-2 OS, WI-38, MRC-5, MDCK,FRhL-2, PERC6, and others. Cells capable of expressing recombinantproteins are known to those skilled in the art, and any of these cellsare encompassed in this invention. In one embodiment, the cell isAV529-19 and the recombinant protein is ICP8 and pUL5.

Methods of Introducing ICP0

ICP0 can be introduced to a cell comprising a nucleic acid encoding arecombinant protein via any method known to those of skill in the art,such as, for example, by transfection or transduction. The ICP0 nucleicacid may be DNA or RNA. Transfection is a process known to those ofskill in the art for introducing nucleic acids into cells. Transfection,as used herein, includes, but is not limited to, lipid transfection,chemical transfection, and physical methods of transfection such aselectroporation. In one embodiment, ICP0 is introduced to a cell viatransfection. In one embodiment, the transfection is lipid transfection.In another embodiment, the transfection is chemical transfection. Instill other embodiments, the transfection is physical transfection, suchas, for example, electroporation. The ICP0 nucleic acid sequence may betransfected into a eukaryotic or prokaryotic cell.

In other embodiments, the ICP0 nucleic acid sequence is introduced intothe cell comprising DNA capable of expressing a recombinant protein viatransduction. Transduction is used herein to comprise introducing DNAinto a cell via a viral vector. The term “viral vector” denotes any formof nucleic acid that is derived from a virus and that is used for thetransfer of genetic material into a cell via transduction. The termencompasses viral vector nucleic acids, such as DNA and RNA,encapsidated forms of these nucleic acids, and viral particles in whichthe viral vector nucleic acids have been packaged. The practice of theinvention is not constrained by the choice of viral vector, as manyforms of viral vectors are known to those skilled in the art. In certainembodiments, the viral vector used for transduction may be retrovirus,lentivirus, adenovirus, adeno-associated virus, or herpesvirus.

Transduction using non-viral vectors is also encompassed. In oneembodiment the non-viral vector is Ormosil (organically modified silicaor silicate).

The expression vector/construct used in the transduction andtransfection methods typically contains a transcription unit orexpression cassette with all the elements required for expression of theICP0 sequence. The particular expression vector that is used totransport the genetic information into the cell may be any conventionalvector used by those of skill in the art. Various means may be used tointroduce ICP0 nucleic acid sequence into host cells, including the useof calcium phosphate transfection, polybrene, protoplast fusion,electroporation, biolistics, liposomes, microinjection, plasma vectors,viral vectors and any of the other well-known methods for introducingcloned genomic DNA, cDNA, synthetic DNA or other foreign geneticmaterial into a host cell. The selection of vector and method oftransfection/transduction is limited only by the need to be capable ofsuccessfully introducing ICP0 into the host cell capable of expressingit. The practice of the invention is also not constrained by the choiceof promoter in the genetic construct. Plasmids may drive expression ofICP0 using inducible or non-inducible promoters. Inducible promoters mayinclude those controlled by tetracycline, doxycycline, IPTG, or othersystems. Non-inducible promoters may include CMV, SV40, CAG, or others.

After the ICP0 is introduced to the cell capable of expressing arecombinant protein, the ICP0 nucleic acid sequence may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid, ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

In other embodiments, the ICP0 is introduced to a cell capable ofexpressing a recombinant protein by introducing a virus that naturallyexpresses ICP0. In one embodiment, ICP0 is introduced by introducingHSV-1, HSV-2, or BHV-1 viruses. HSV-1, HSV-2, and BHV-1 viruses expressthe ICP0 protein as shown in Sequence IDs 4-6, respectively. In oneembodiment, infection of cells capable of expressing a recombinantprotein with a virus that encodes ICP0 would lead to increased ICP0levels. In certain embodiments, the HSV-1, HSV-2, or BHV-1 viruses maybe used to introduce ICP0. In one embodiment, replication defectiveviruses, such as HSV529 or other replication defective HSV-2 viruses,may be used to express ICP0.

In certain embodiments, ICP0 may already be expressed in the cell at thetime that a recombinant protein is introduced. In other embodiments,ICP0 is introduced into a cell that does not normally express it.

Methods for Producing Replication Defective Virus In Vitro

In one embodiment, methods for producing, and methods for increasing theyield, of a replication defective virus in vitro are encompassed. Inthese methods, ICP0 is provided to a cell comprising at least onerecombinant protein, wherein the recombinant protein is a protein thatis defective in the replication defective virus. A replication defectivevirus is provided to the cell at the same time as, or after, ICP0. ICP0increases the expression of the recombinant protein, thereby providingthe recombinant protein in trans to the replication defective virus. Thereplication defective virus is thus capable of replicating, and the invitro cell system produces and increases the yield of the replicationdefective virus.

a. Production of HSV-2 Vaccine HSV529

In one exemplary embodiment, the replication defective virus is HSV529.As previously described (See M. C. Bernard et al., PLOS One10(4):e0121518 (2015)), HSV529 lacks two viral DNA replication genes(UL5 and UL29) and is generated using the complementary helper cellline, AV529-19. AV529-19 is a Vero cell line (ATCC, CCL-81.2), which hasbeen stably transfected to supply the UL5 and ICP8 HSV-2 proteins intrans. Infection of AV529-19 cells by HSV529 can be performed followingpublished protocols (See, S. T. Mundle et al., PLoS One 8, e57224(2013). Briefly, AV529-19 cells are grown to confluence, and theninoculated with HSV529 at a multiplicity of infection (MOI) of 0.01 ininfection medium (40% OptiPro in DPBS with 0.5× cholesterol, 50 mMsucrose, for example). Infection can proceed over time, such as beingallowed to proceed at 34° C. for approximately 72 hr.

In one embodiment, ICP0 is introduced to AV529-19 cells prior toinfection with HSV529 in order to increase expression of proteins suchas ICP8 and pUL5. In certain embodiments, ICP8 and pUL5 may be those ofHSV-2. In other embodiments, ICP8 and pUL5 may come from differentsources such as HSV-1 or BHV-1. In certain embodiments, ICP0 can beintroduced by transfection. In certain embodiments, ICP0 can beintroduced via transduction. In certain embodiments, AV529-19 cellsexpressing ICP0 can then be infected with HSV529, leading to anincreased yield of the viral vaccine. In one embodiment, an increasedyield is a yield that quantitatively is an amount of virus obtainedfollowing use of AV529-19 cells expressing ICP0 that is greater than theyield following use of control AV529-19 cells that do not express ICP0.

In certain embodiments, ICP0 is introduced at different time pointsprior to HSV529 infection. In one embodiment ICP0 is introduced 10, 9,8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.4, 0.3, 0.2 or 0.1 hours prior tointroducing the replication defective virus. In certain embodiments, theMOI of infection and time to harvest are optimized with the protocolincluding ICP0 introduction to ensure maximal production of HSV529. Incertain embodiments, the levels of ICP8 and/or pUL5 are monitoredfollowing expression of ICP0 to aid in optimizing the timing of ICP0introduction.

Various means of viral purification of HSV529 have been described,including ultracentrifugation and sucrose cushion-based purification, aswell as chromatography-based purification (See, S. T. Mundle et al.,PLoS One 8, e57224 (2013). In some embodiments the replication defectivevirus produced after ICP0 is introduced is purified, such as byultracentrifugation or cesium chloride purification. In certainembodiments, mechanical cell disruption or chemical elution of HSV529from the surface of the infected AV529-19 cells can first be performed,with evidence that chemical elution may allow higher purity viralpurification. In one embodiment, purification of the virus may beperformed by a combination of dextran sulfate elution followed byBenzonase treatment, depth filtration, anion exchange chromatography,and ultrafiltration/diafiltration. In one embodiment, the virusisolation process follows GMP protocols and produces vaccine preparationto be used for clinical investigation, such as HSV529. In oneembodiment, the virus purification process follows GMP protocols and isa prophylactic therapy for infection with HSV-2.

Methods for Increasing Expression of Recombinant Proteins In Vitro

Methods for increasing the expression of recombinant proteins in vitroare encompassed. In one embodiment, the recombinant protein is anantibody or a vaccine antigen. In other embodiments, the recombinantprotein is a hormone, enzyme, or other mammalian protein. In otherembodiments, the recombinant protein is a peptide used for imaging,diagnostic, or therapeutic purposes. In other embodiments, therecombinant protein is an antibody. Methods for increasing theexpression of recombinant proteins in vitro comprise introducing ICP0 toa cell capable of expressing a recombinant protein and isolating therecombinant protein, wherein the recombinant protein is increased incells introduced with ICP0 as compared to cells that have not beenintroduced to ICP0.

Herpes Simplex Virus Infected Cell Polypeptide Zero (“ICP0”)

The ICP0 of the invention may be any ICP0 from a herpes simplex virus(HSV), including, but not limited to, ICP0 from HSV-1, HSV-2, or BHV-1.In one embodiment an HSV-1 ICP0 is encompassed. In another embodiment,HSV-2 ICP0 is encompassed. In yet other embodiments, bovine BHV-1 ICP0is encompassed.

In certain embodiments, the ICP0 envisioned is any protein having a RINGfinger domain, and having ubiquitin ligase activity.

The RING finger domain is known to those of skill in the art and is asdescribed in Barlow, J. Mol. Biol. (1994) 237, 201-211 and Boutell andEverett, J. Gen. Virol (2013) 94:465-481 at FIG. 1. The skilled artisancan determine by sequence analysis whether any candidate ICP0 has a RINGfinger domain.

The skilled artisan can also determine whether any candidate ICP0 hasubiquitin ligase activity, by, for example, conducting a “UbiquitinLigase Activity Assay” (see, e.g. Boutell et al (January 2002) JVirology. 76(2):841-50). If a candidate ICP0 exhibits E3 ubiquitinligase activity in vitro, as evidenced by ubiquitin chain formation, theICP0 candidate satisfies the “Ubiquitin Ligase Activity Test.”Similarly, if a candidate ICP0 lacks ubiquitin ligase activity in vitro,said ICP0 fails to satisfy the “Ubiquitin Ligase Activity Test.”

“Ubiquitin Ligase Activity Assay”: ICP0, as an E3 ubiquitin ligase,interacts with both E1 and E2 ubiquitination conjugating enzymes tocatalyze the formation of poly-ubiquitination chains on substrateproteins. Thus, a Ubiquitin Ligase Activity Assay determines whether acandidate protein can catalyze the formation of poly-ubiquitinationchains on substrate proteins in the presence of E1 and E2. A sampleactivity assay is described below. See, also, detailed methods inBoutell and Davido, Methods (2015) 51046-2023(15):00150-4, and Everettet al. (April 2010) J Virol 84(7):3476-87.

Reaction Mixture Components:

a. E1 ubiquitin-activating enzyme, which can be purified frombaculovirus-infected cell extracts or, alternatively, the enzyme can beexpressed with an N-terminal polyhistidine tag by a recombinantbaculovirus followed by purification from extracts by nickel affinitychromatography. For example, the E1 enzyme can be purified from HeLacell S100 extracts using ubiquitin affinity chromatography, as describedin Boutell 2002, or polyhistidine-tagged E1 enzyme can be expressed andpurified by nickel affinity chromatography, as described in Everett2010.

b. E2 ubiquitination conjugating enzymes can be expressed in recombinantEscherichia coli as a polyhistidine or GST fusion-proteins and purifiedby nickel or glutathione affinity chromatography. Examples ofpolyhistidine-tagged UBE2D1 (UbcH5a) have been described in theliterature (Boutell 2015, Everett 2010) that can be used in theUbiquitin Ligase Activity Test.

c. Wild-type ubiquitin (such as from Sigma Aldrich; U6253)

Exemplary Ubiquitin Ligase Activity Test: An example ubiquitinationassay is a final reaction volume of 10 μl in 50 mM Tris (pH 7.5), 50 mMNaCl, 1 mM MgCl2, and 5 mM ATP (Sigma-Aldrich; A7699) supplemented with10 ng E1 and 40 ng of E2 (UBE2D1). To this mixture, 90 ng of candidatepurified ICP0 protein is added (Boutell 2015; with a similar reactiondescribed in Everett 2010). The reaction is activated by addition of 1μg of wild-type (Sigma-Aldrich; U6253) or methylated (BostonBiochem;U-501) ubiquitin per reaction and incubated at 37° C. for a time range,for example over 0-90 min Assays can be terminated by addition of3×SDS-PAGE loading buffer supplemented with 8M urea and 100 mMdithiothreitol (DTT), followed by heat denaturation at 95° C. for 10min, and then SDS-PAGE (12% Bis-Tris NuPAGE; Life Technologies).Following transfer to nitrocellulose membranes, membranes can be blockedin PBS with 10% fetal calf serum for 1 h at room temperature. Blots canthen be incubated with anti-ubiquitin antibody (e.g., P4D1 [1/1000]),followed by incubation with appropriate secondary antibodies, based onthe preferred quantification analysis system. Quantification ofubiquitination can be performed using these membranes, such as bynear-IR imaging using an Odyssey CLx infrared imaging system (LI-CORBiosciences, Boutell 2015) at a resolution of 84 mm or by enhancedchemiluminescence reagent (NEN, Boutell 2002) and exposure to filmProduction of polyubiquitin chains can be measured using quantificationof the labeled ubiquitin. For example, ubiquitin levels after a 1 hreaction can be measured (Everett 2010) or a time-course of ubiquitinlevels over a 90-minute period can be measured (Boutell 2015).

In one embodiment the ICP0 is identical to the amino acids of SEQ ID NO:4, SEQ ID NO: 5, or SEQ ID NO: 6. The ICP0 may be 99, 98, 97, 96, 95,94, 93, 92, 91, 90, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73,72, 71, or 70% identical to any of SEQ ID NO: 4, SEQ ID NO:5, or SEQ IDNO:6, so long as the ICP0 maintains ICP0 activity in the UbiquitinLigase Activity Assay.

Stable Cell Lines Expressing ICP0 for Increasing Expression ofRecombinant Proteins

In one embodiment, methods for generating stable lines expressing ICP0are encompassed. In certain embodiments, a plasmid containing ICP0 istransfected into a cell line, such as HEK, CHO, HeLa or others, and astable cell line expressing ICP0 is generated using an appropriateselection method, such as, for example, antibiotic resistance. Clones ofthe ICP0 stably-transfected line are frozen and then the line ismaintained under selection for stable ICP0 expression. In oneembodiment, a method for increasing the production of a recombinantprotein in the stably-transfected cell is encompassed, comprisingmaintaining the stably-transfected cell line in culture, and introducinga nucleic acid comprising a recombinant protein, wherein the recombinantprotein is expressed at higher levels than in cells that do not compriseICP0. In another embodiment, a cell line stably-expressing ICP0 line isused to screen for recombinant proteins whose production can beincreased by co-expression with ICP0.

Complementary Cell Lines Stably-Expressing ICP0 for IncreasingExpression of Replication Defective Viruses

In certain embodiments, methods for generating a complementary cell linestably transfected with ICP0 for production of a replication defectiveviral vaccine is encompassed. In one embodiment, the complementary cellline stably transfected with ICP0 is the AV529-19 cell line, and thereplication defective viral vaccine is HSV529. In certain embodiments,stable transfection of ICP0 in the complementary cell system increasesthe yield of the replication defective virus after infection of thecells compared with the control cell system that does not express ICP0.

EXAMPLES Example 1. HSV529 Infection of the Cell Line AV529-19 InducesTranscription and Expression of ICP8

The vaccine HSV529 is a replication defective vaccine strain of herpessimplex virus 2 (HSV-2) missing two genes, UL5 and UL29. HSV529 and itscomplementing cell line AV529-19 have been described previously. See, S.T. Mundle et al., PLoS One 8, e57224 (2013); S. Delagrave et al., PLoSOne 7, e46714 (2012).

The UL5 and UL29 genes are necessary for replication of HSV529, andtheir deletion results in a virus that can deliver viral genomic DNAinto human cells and elicit an immune response against the viralproteins, but cannot replicate. The inability to replicate results in avaccine that is safe to the vaccine recipient. HSV529 can not replicatein partially complementing cells, i.e., HSV529 does not grow if only oneof UL5 and UL29 genes are expressed (FIG. 1). HSV529 was not able togrow in either V827 cells (expressing only UL29 and not UL5) or L2-5cells (expressing only UL5 and not UL29); however, growth was seen inAV529-19 cells which express both complementing genes.

The complementing cell line AV529-19 is a Vero cell line into whichHSV-1 orthologs of the two deleted genes, UL5 and UL29, were stablyintegrated. AV529-19 cells provide HSV529 with the missing proteins pUL5(helicase encoded by UL5, where ‘p’ denotes the polypeptide product ofthe gene UL5) and ICP8, respectively, in trans. See, A. Azizi et al., JBiotechnol 168, 382 (December 2013). Because a CMV promoter drives theexpression of UL5 and UL29, it was expected that both genes would beconstitutively expressed in AV529-19 cells, and that upon infection withHSV529, the viral vaccine genome would be replicated and new progenyvirions comprising the HSV529 genome would be produced by thecomplementing AV529-19 cells.

Surprisingly, however, it was observed that complementation of HSV529 inAV529-19 cells yields up to 12-fold less virus per cell than whenAV529-19 cells are infected with wild type HSV-2 (FIG. 2). As part of aneffort to understand the basis for the imperfect complementation, theexpression of ICP8 was studied. Antibodies appropriate for western blotdetection of ICP8 were available. Expression of ICP8 was undetectable inuninfected AV529-19 cells, despite being regulated by a constitutivepromoter, and despite the clear ability of the cells to complementHSV529 (FIG. 3). Immunodetection with a mouse anti-HSV-1-ICP8 antibody(Abcam, ab20194) indicated that HSV529 infection in AV529-19 cells leadsto readily detectable ICP8 expression at 41 hours post-infection (toppanel of FIG. 3). No expression of ICP8 was observed in mock-infected(NC) cells. The housekeeping gene product

GAPDH was not affected by HSV529 infection (mouse anti-GAPDH; SantaCruz, sc32233) as shown in middle panel of FIG. 3. Viral proteinexpression was only seen in infected HSV529 cells using immunodetectionwith rabbit anti-HSV-2 antibody (Abcam, ab9534) as shown in the bottomblot of FIG. 3.

We observed that ICP8 could be detected only after cells were infectedwith HSV529, but not before (FIG. 3 & FIG. 4).

Example 2. HSV529 Infection of the Cell Line AV529-19 IncreasesExpression of ICP8 Encoded by a Replicon System

Using a Venezuelan equine encephalitis (VEE) replicon system, theexpression of a replicon-encoded ICP8 in Vero cells after infection withHSV529 was tested (FIG. 4). The UL29 gene was cloned into plasmidp5′VEErep/GFP/Pac, described in Petrakova et al., J Virol 79, 7597 June2005), replacing the GFP ORF with the UL29 ORF. In the VEE replicons,which is an RNA episome, expression of UL29 or UL5 is driven by thesubgenomic VEE promoter encoded by the plasmid p5′VEErep/GFP/PacReplicon RNA was produced by in vitro transcription of the plasmiddriven by an SP6 promoter. The resulting UL29 replicon RNA wastransfected into the Vero cells by electroporation using a BioRad GenePulser II (320V, 950 μF), and the transfected cells were placed underselection using puromycin (10 μg/mL) to ensure retention of thereplicon. A similar replicon encoding UL5 was done in parallel as acontrol. Cell samples were taken 0, 4, 8 or 24 hours post-infection withHSV529, and tested for ICP8 expression by western blot (FIG. 4). Theanti-HSV 1 ICP8 rabbit serum 3-83 was used as a primary antibody and agoat polyclonal anti-rabbit labeled with horseradish peroxidase as asecondary. The SuperSignal West Pico chemiluminescence substrate wasused for detection.

ICP8 was not detected at the earliest time points (FIGS. 4: 0 h and 4 htime points). Bands of the expected molecular weight (>100 kD) wereobserved at 24 h for the UL29 replicon cell line, and at both 8 h and 24h in the AV529-19 cells. As expected, UL5 cells do not produce ICP8 atany time point even with HSV529 infection. Unexpectedly, ICP8 expressionwas induced when expressed from a replicon. The increased expression ofICP8 appears to be mediated by HSV529 even when ICP8 is expressed froman RNA molecule (the replicon). Therefore, the ability of HSV529 toincrease expression of a gene of interest, in this case ICP8, is notdependent on the gene being encoded by DNA.

Example 3. HSV529 Infection of the Cell Line AV529-19 Increases theAmount of UL29 Transcript in Cells

To further our understanding of the increase in ICP8 expression inHSV529-infected cells expressing recombinant UL29, we tested whether acorresponding increase in UL29 mRNA could be detected after infection. Aquantitative reverse transcription polymerase chain reaction (qRT-PCR)targeting the 3′ untranslated region (UTR) of the recombinant UL29 mRNAusing the forward primer SEQ ID1; the reverse primer SEQ ID2; and theprobe SEQ ID3; as shown in Table 2 was implemented. The sequence used togenerate these primers is exclusively associated with expression of therecombinant UL29 gene. The UL5 gene, for example, is expressed using adifferent vector having a different 3′ UTR and, therefore, will not betargeted. AV529-19 or unmodified Vero cells were infected with eitherHSV-2 (strain 186 syn+1) or HSV529 (FIG. 5). As expected, unmodifiedVero cells had undetectable amounts of recombinant UL29 mRNA at all timepoints, while AV529-19 had detectable but very low amounts of therecombinant UL29 mRNA at 0 and 6 h post-infection. The mRNAconcentration increased by 22- and 26-fold following infection for theHSV529 and HSV-2-infected cells, respectively. UL29 mRNA inductionpeaked at 24 h post-infection in HSV-2-infected cells and at 48 hpost-infection in HSV529-infected cells. These results indicate that theintracellular concentration of the UL29 transcript is increased byinfection of cells with either wild-type HSV-2 or HSV529. The kineticsof increased UL29 mRNA expression are consistent with the observedincrease in protein levels of ICP8, the UL29 gene product.

TABLE 2 Description of the Primers Used forTaqMan Quantification of Recombinant UL29 mRNA Forward Reverse AmpliconTarget Description Primer Primer Probe length UL29 3′UTR GCCAG GGGAGCCCCG 62 bp sequence CCATC TGGCA TGCCT of pcDNA3.1 TGTTG CCTTCC TCCTTused to TTTGC A (SEQ ID transfect (SEQ ID (SEQ ID NO: 3) AV529-19 NO: 1)NO: 2)

Example 4. ICP0 Increases the Expression of ICP8 in the ComplementingCell Line AV529-19

We hypothesized that an immediate early (IE) protein of HSV-2 might beresponsible for the induction of UL29 expression. Therefore, a panel ofadenovirus vectors expressing the 5 HSV-2 IE proteins: ICP0, ICP4, ICP22(C-terminal fragment), ICP27 (combination of N-terminal and C-terminalfragments), and ICP47 were tested. AV529-19 cells were transduced at anMOI of 20 with these various vectors and harvested 24 h later forwestern blotting using an anti-ICP8 antibody (FIG. 6). A single bandshowing expression of ICP8 was seen only in the cells transduced withthe ICP0-expressing vector. None of the other adenovirus vectorsexpressing other HSV-s IE proteins showed any effect on ICP8 expression.The ‘empty’ adenovirus vector itself also caused no expression of ICP8.This result strongly suggests that the induction of ICP8 observed inExamples 1-3 is due to the protein ICP0 expressed by both HSV-2 andHSV529 following infection of AV529-19.

In a control experiment, transduction of the ICP0-expressing adenovirusresulted in expression of ICP8 in AV529-19 cells, but not in HEK293Acells (FIG. 7). HEK293A cells, a complementing cell line used to produceadenovirus vectors, and the AV529-19 cell line were infected with anadenovirus vector expressing ICP0. Western blot analysis at 24 h afterinfection indicated that ICP0 is not detectable in AV529-19 cells,presumably because its expression level was below the detection limit ofthe polyclonal antibody. Immunostaining of the ICP8 band in the AV529-19cells appeared to have strengthened, which is consistent with thespecificity of the polyclonal antibody, while no signal was seen inHEK293A cells. These results indicate that ICP0 expression does notresult in ICP8 expression in cells that are not capable of expressingICP8, such as HEK293A cells.

Example 5. ICP0 Increases the Expression of Episomally-EncodedRecombinant Proteins, as Well as of Proteins of Non-Viral Origin

Whether ICP0 could enhance the expression of recombinant proteins otherthan ICP8 was tested, as was whether this enhancement could be observedusing recombinant genes carried on episomal DNA molecules rather than onthe host chromosomal DNA. Cells were co-transduced with adenovirusvectors (MOI of 20) expressing various proteins and, 24 hours later,with either an empty adenovirus vector or an adenovirus vectorexpressing ICP0 (MOI of 20). In this experiment, the recombinantproteins of interest were HSV-2 proteins gD313, VP16, and fireflyluciferase. Although AV529-19 cells were used, other cell lines may beused instead (as will be shown in later Examples). Twenty-four hoursafter co-transduction, infected cells were analyzed on western blotsusing commercially available antibodies (FIG. 8A and FIG. 8B).Remarkably, in each case, expression of co-transduced proteins wasincreased by ICP0 but not by the empty adenovirus vector (results for gDshown in FIG. 8A and results for VP16 and firefly luciferase shown inFIG. 8B). These results show that not only is the expression of HSV-2proteins (ICP8, gD, and VP16) increased by ICP0, but so is theexpression of a eukaryotic enzyme (firefly luciferase). Moreover, ICP0increases expression of episomally-encoded proteins, in addition to theincreases of chromosomally-encoded ICP8 shown in earlier Examples.

To investigate whether the induced expression of recombinant proteins byICP0 was specific or due to a global effect on protein expression in thecell, the expression of the housekeeping gene GAPDH was analyzed bywestern blotting of cells co-transduced with a recombinant protein andwith either ICP0 or a negative control adenovirus vector (FIG. 9A). Noobvious changes in the expression of this protein were detected as aresult of ICP0 expression. We also used SDS-PAGE and Simple-Bluestaining analysis of a subset of the same samples for a broader view ofprotein expression in these cells (FIG. 9B). Negative control (NC)un-transduced cells, empty vector (AdV) cells, and cells co-transducedwith gD or VP16 show a similar pattern of bands. Therefore, the effectof ICP0 on protein expression appears to be specific to the recombinantproteins or to a small subset of cellular proteins that includes therecombinant proteins.

Example 6. ICP0 Increases the Expression of Recombinant Proteins Encodedby Different Plasmids and Using Different Promoters

In the above Examples, the recombinant proteins were encoded by a stablyintegrated CMV promoter-driven transgene (ICP8) or by an adenovirusvector that also drives expression using the CMV promoter. We thereforetested the ability of ICP0 to increase the expression of a recombinantprotein encoded by two different plasmids driving the expression of thetransgene with either the CMV promoter or the SV40 promoter. ICP0transduction increased expression of firefly luciferase by 2.8-fold and5.1-fold for plasmids with the SV40 and CMV promoters, respectively,compared with control vector transduction (FIG. 10). Therefore,ICP0-mediated enhancement of recombinant, plasmid-borne gene expressionis independent of the promoter used.

The data shown in FIG. 10 were obtained using AV529-19 cells. As statedabove, these are Vero cells engineered with chromosomally integratedcopies of the genes UL29 and UL5. To show that UL29 and UL5 are notnecessary for the observed effect of ICP0, we reproduced part of theexperiment using Vero cells (FIG. 11). As seen in FIG. 11, ICP0transduction produced a 9-fold increase in luciferase expression in Verocells, confirming that the enhancing effect of ICP0 can be achievedwithout expression of UL29 or UL5. The difference between controladenovirus vector and vector expressing ICP0 was extremely statisticallysignificant by ANOVA analysis (P<0.0001).

To confirm that the ICP0 polypeptide is responsible for the enhancedexpression effect, the ability of two different ICP0 mutants to induceICP8 expression was assessed. Wild-type and mutant ICP0 were deliveredto AV529-19 cells via an adenovirus vector. Western blotting with arabbit polyclonal anti-HSV-2 antibody that cross-reacts with HSV-1 ICP8clearly shows the expression of ICP8 in wild-type ICP0-treated cells,but not in cells treated with a version of ICP0 in which the N-terminal⅓ was deleted (⅔ ICP0 C-H construct), or another version in which about5% of amino acids in the protein are substituted (ICP0 5% mut-H) (FIG.12). Therefore, the ICP0 polypeptide sequence must be mostly preservedto increase expression of ICP8 in AV529-18 cells.

Example 7. ICP0 Increases Recombinant Protein Expression when it isProvided to Cells Via a Plasmid

In the above Examples, ICP0 was expressed using either HSV-2, HSV529, oran adenovirus vector. We therefore tested the ability of ICP0 expressedfrom a plasmid to increase protein expression. AV529-19 cells weretransiently transfected with an empty plasmid or with ICP0 expressedfrom a CMV promoter in two different plasmids (pcDNA3.1 and 0603), andthen levels of expression of ICP8 were measured by western blot.Expression of ICP8 was clearly induced by ICP0 transfection by eitherplasmid in cells harvested 42 h post-transfection (FIG. 13). The topblot of FIG. 13 was probed with the rabbit anti-HSV-2 polyclonalantibody which cross-reacts with HSV-1 ICP8 (red arrow). Induction ofICP8 was clearly detected in the AdICP0 lane as well as in cellstransfected with ICP0 expressed from a plasmid. To confirm the identityof ICP8 in the cells, another western blot was carried out with the samesamples and probed with a mouse anti-HSV-1 ICP8 monoclonal antibody andshowed similar results (lower blot of FIG. 13). The ICP0 expressed frompcDNA3.1 bears a C-terminal poly-histidine tag, and this tag did nothinder ICP8 induction. Control samples, either with no transfection (NC)or in which an empty vector (pcDNA) was transfected, showed no ICP8expression.

While transfection of plasmids containing ICP0 DNA was clearly capableof inducing ICP8, this induction was weaker than that which was mediatedby transduction with an adenovirus vector expressing ICP0 (FIG. 13). Wehypothesized that the lower efficiency of transient transfectioncompared to adenovirus-mediated transduction may explain thisobservation. To rule out the possibility that adenovirus components hadincreased the efficiency of the ICP0 enhancement, we transfectedAV529-19 cells with ICP0 plasmids and 24 hours later transduced thetransfected cells with ‘empty’ adenovirus vector. Cells were thenassayed 36 h later for ICP8 protein expression. Adenovirus vectortransduction following plasmid transfection did not enhance ICP8 proteinexpression (FIG. 14). These results suggest that lower ICP0 plasmidtransfection efficiency limited the enhancement in protein expressionfor cells transfected with plasmid compared to cells transduced withadenovirus.

Example 8. ICP0 Increases the Yield of HSV529

As discussed in Examples 1 and 3, HSV-2 replicated more efficiently inAV529-19 cells than does HSV529 (FIG. 2). The data also indicated thatHSV529 infection causes a slower and slightly lower increase in theexpression of the recombinant UL29 transcript compared to HSV-2 (FIG.5). Given this correlation between ICP8 expression and viral yields, wehypothesized that AV529-19 cells expressing ICP0 prior to infection withHSV529 would induce ICP8 expression and would therefore complement thevirus more efficiently and produce higher yields when infected withHSV529. Transduction of AV529-19 with adenovirus vector expressing ICP0before infection with HSV529 showed a clear increase in HSV529 yieldscompared to transduction with an empty adenovirus vector (FIG. 15).Transduction with ICP0 was carried out at 1 h, 4 h, or 22 h prior toHSV529 infection and, compared to the yields obtained with empty vector,resulted in increases of 2.8-, 4.3-, and 2.2-fold, respectively.Compared to a baseline, un-transduced sample of HSV529-infected cells(HSV529 only), the greatest enhancement in yield (5.5-fold) was achievedby infection of HSV529 at 1 h following transduction of adenovirusexpressing ICP0. Thus, appropriately timed expression of recombinantICP0 can trigger increased production of the vaccine HSV529 in AV529-19cells.

Example 9. Inducible Expression of ICP8

Whether it is possible to induce the expression of ICP8 in AV529 cellsusing a small molecule compound was tested. The TetOne vector was usedto construct a plasmid that expresses ICP0 under the control of adoxycycline-sensitive promoter (TetOne-ICP0). This plasmid wastransfected into the AV529 cell line. Increasing the concentration ofdoxycycline in the culture medium of transfected cells induced theexpression of the protein ICP8, which is encoded by the stably insertedUL29 gene in this line (FIG. 16). Similar results were seen for anengineered version of the plasmid to which a polyhistidine tag wasappended at the carboxyl terminus (TetOne-ICP0 his). Control AV529 cellstransfected with an empty TetOne vector did not show increasedexpression of ICP8. Therefore, these data show that ICP8 expression canbe induced, by a mechanism of induced expression of ICP0, using a smallmolecule drug.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the embodiments. The foregoingdescription and Examples detail certain embodiments and describes thebest mode contemplated by the inventors. It will be appreciated,however, that no matter how detailed the foregoing may appear in text,the embodiment may be practiced in many ways and should be construed inaccordance with the appended claims and any equivalents thereof.

As used herein, the term about refers to a numeric value, including, forexample, whole numbers, fractions, and percentages, whether or notexplicitly indicated. The term about generally refers to a range ofnumerical values (e.g., +/−5-10% of the recited range) that one ofordinary skill in the art would consider equivalent to the recited value(e.g., having the same function or result). When terms such as at leastand about precede a list of numerical values or ranges, the terms modifyall of the values or ranges provided in the list. In some instances, theterm about may include numerical values that are rounded to the nearestsignificant figure.

What is claimed is:
 1. A method for producing a replication defectiveHerpes simplex virus (HSV) in cell culture comprising: a) providing acell comprising a nucleic acid encoding at least one recombinantprotein, wherein the recombinant protein is required for replication ofsaid replication defective HSV; b) introducing a herpes simplex virusinfected cell polypeptide zero (ICP0) to the cell by transduction with avirus other than HSV or transfection, thereby increasing the expressionof the recombinant protein; c) introducing a replication defective HSVto the cell, wherein the recombinant protein complements the replicationdefective virus in trans; d) replicating said replication defective HSVin the cell; and e) isolating the replication defective HSV from thecell; wherein the recombinant protein is ICP8 and/or pUL5.
 2. A methodfor producing replication-defective Herpes simplex virus 2 (HSV-2)vaccine HSV529 comprising the following steps, in order: a) introducingICP0 to AV529-19 cells, wherein ICP0 increases the expression of atleast one of ICP8 or pUL5 in the AV529-19 cells; b) infecting theAV529-19 cells with HSV529; c) replicating HSV529 in the cell; and d)isolating HSV529 from the cell.
 3. A method for increasing the yield ofreplication defective HSV-2 vaccine HSV529 comprising the followingsteps, in order: a) introducing ICP0 to AV529-19 cells, wherein ICP0increases the expression of at least one of ICP8 or pUL5 in the AV529-19cells; b) infecting the AV529-19 cells with HSV529; c) replicatingHSV529 in the cell; and d) isolating HSV529 from the cell.
 4. The methodof claim 1, wherein the cell is AV529-19.
 5. The method of claim 1,wherein the recombinant protein is ICP8 or pUL5.
 6. The method of claim1, wherein the recombinant protein is viral or non-viral.
 7. The methodof claim 1, wherein the cell is a mammalian cell.
 8. The method of claim1, wherein the cell is selected from Vero, BHK, CHO, HKB, HEK, NSO,WI-38, MRC-5, MDCK, or FRhL-2.
 9. The method of claim 1, wherein ICP0 isintroduced by transduction.
 10. The method of claim 9, wherein ICP0 isintroduced by transduction with an adenovirus expressing ICP0.
 11. Themethod of claim 1, wherein ICP0 is introduced by transfection.
 12. Themethod of claim 1, wherein the ICP0 is introduced by transfection with aplasmid encoding ICP0.
 13. The method of claim 12, wherein the plasmidcomprises an inducible or non-inducible promoter driving expression ofICP0.
 14. The method of claim 13, wherein the inducible promoter isselected from the group consisting of chemically- orphysically-regulated promoters.
 15. The method of claim 13, wherein thenon-inducible promoter is a non-inducible CMV promoter or anon-inducible SV40 promoter.
 16. The method of claim 13, wherein thenon-inducible promoter comprises a TATA box, a GC-box, a CCAAT box, a Brecognition element, and/or an initiator element.